Pili have been shown to be important adhesive organelles for many bacterial pathogens that cause diarrheal and urinary tract disease. We have recently identified a gene in Vibrio cholerae, which we have named tcpA, that encodes a major pilus subunit. Transposon insertions in tcpA virtually abolish virulence and dramatically decrease colonization in animal models. The TcpA pilin shares extensive homology with the N-methylphenylalanine pilins that are produced by a diverse group of bacterial pathogens. While this class of pili are potential vaccine targets against several important infectious diseases there is little known about their biology. Genetic studies of other pilus classes have revealed information critical for understanding pilus function and for intelligent vaccine development. For example, in the case of all pili types of E. coli that have been studied, the pilus is composed of multiple components that are encoded by a series of adjacent genes. For several pilus types the functional adhesin is distinct from the pilin. In order to determine how the TcpA nd related pili function we plan to further characterize the tcpA locus both genetically and biochemically. First, the tcpA gene will be defined at the level of its DNA sequence. Knowledge of this sequence will be used to construct a series of precise, nonpolar tcpA deletion mutations which will be recombined into the V. cholerae chromosome and tested for their effects on virulence in animal models. These studies will be extended by cloning a substantial region of the chromosome adjacent to the tcpA structural gene and determining, by mutation, if the products of other genes contribute to TcpA pilus function. This analysis will determine if the TcpA pilin protein is itself sufficient to promote colonization or if additional gene products are required. The organization of such genes and the identification of their products will be determined.